How to perform specificity during method validations?

 Specificity is one of the method validation parameter. Specificity defined by ICH Q2(R1), is as below,

“Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.”

So in the context of specificity, the interested component must get explicitly assessed in presence of other sample components. To explain it further, let us take an example of a finished product such as tablets. Test solution of tablets consists of interested compounds (such as API and impurities) as well as the components which are of no interest (such as excipients). The excipients, though they are of no interest, one can’t completely remove them from test solution and hence it is important to assess their impact on quantification of analyte. Also, in case of assay analysis, the impurities will become the components of no interest and one must assess impurity interference while performing specificity.

Let us understand how specificity is conducted for various tests such as assay, related substances, dissolution and identification. 

A.    Specificity for Assay:

In case of assay, specificity must be studied for blank/diluent, placebo (in case of finished product such as tablets/capsule/cream etc.), impurity and degradation product (forced degradation). Let us discuss one by one in detail

i. Blank/Diluent Interference: Diluent may interfere during quantification of analyte. To nullify diluent interference- blank measurement is generally carried out during titrimetric or potentiometric analysis. In case of spectroscopic technique – the baseline is adjusted to zero with diluent. However, in case of chromatographic techniques, such as HPLC or GC, the diluent interference may not be removed fully just by doing auto-zero. The components or impurities present in diluent may get retained on column and can interfere with analyte. And hence it is necessary to assess diluent interference for assay method with the chromatographic technique such as HPLC, GC etc. You must aim for no interference from diluent during quantification of analyte.

ii. Placebo interference: Evaluation of placebo interference is applicable only for drug product such as tablet, capsule etc. Placebo consists of all excipients present in drug product. The excipients may interfere with analyte if they produce response (such as peak area) and elute at the RT of analyte. But placebo will not harm specificity if it does not produce response. So, you will not see any interference from excipient even eluting at the RT of analyte if excipient does not produce response. To evaluate specificity, placebo solution can be prepared by following test preparation, but by omitting API. Measure the response from placebo solution and conclude whether requirement is met. No interference from placebo must be observed in quantification of analyte.

iii. Impurity interference: You need to demonstrate the non-interference of impurities in quantification of analyte. Prepare individual impurity solution for identification. Further, spike impurities at limit level into test solution and assess the interference of impurities at the RT of analyte for HPLC/GC technique. To assess the interference, the peak purity of analyte in the ‘impurity spiked test solution’ can be assessed. The peak purity can be assessed only for UV detector. Peak purity can’t be assessed for GC and other than UV detectors for HPLC. Alternatively, if peak purity can’t be assessed, the assay values with and without impurities present in test solution can be compared to conclude if impurity is interfering with quantification of analyte. The assay difference of less than 2% is generally acceptable.

iv. Forced degradation: Demonstrate the non-interference of degradation products in quantification of analyte. Conduct the forced degradation studies to obtain degraded samples with 5% to 20% degradation wherever possible. Preferably, the following stress conditions are recommended for specificity study. However, stress condition can be decided based on experimental data, or physical properties (Literature data) of the analyte.    Assess peak purity of analyte wherever possible.

Recommended stress conditions:

Heat: At 105°C for about 12 hours.      

Humidity: About 90% RH at 25°C for NLT 7days.     

UV Light: NLT 200 Watt hours/m2    

Fluorescent light: NLT 1.2 million lux hours

Acid: 0.1N HCl refluxed for 30 minutes at 60°C.             

Base: 0.1N NaOH refluxed for 30 minutes at 60°C.

Peroxide: 1% H2O2 refluxed for 30 minutes at 60°C.

Water: Reflux for 6 hours at 60°C.

B. Specificity for Related Substances:

In case of related substances, specificity must be studied for blank/diluent, placebo (in case of finished product such as tablets/capsule/cream etc.), impurity and degradation product (forced degradation). Let us discuss one by one in detail

i. Blank/Diluent Interference: Conduct interference due to diluent in case of chromatographic technique such as HPLC, GC. Inject diluent and evaluate interference at the RT of API and impurities. There must not be any interference due to diluent.

ii. Placebo interference: Establish the non-interference or extent of interference of placebo in estimation of % of impurities. Prepare a placebo solution by following the procedure for test solution using equivalent weight of placebo in portion of test preparation. Analyse placebo solution following test conditions, record the response from placebo and measure the interference at the impurity and API. No interference from placebo must be observed at the RT of impurities and API.

iii. Impurity interference: Prepare individual solution of each impurity and evaluate retention time. Similarly spike all known impurities into test solution at specification level and evaluate peak purity and interference. The peak purity of each impurity must get pass and there shall be no interference from impurity and API to each other. The peak purity can be assessed only for UV detector. Peak purity can’t be assessed for GC and other than UV detectors for HPLC.

iv. Forced degradation: Demonstrate the effective separation of analyte from the degradation product and peaks if any due to components of placebo. Evaluate peak purity for impurity and API. For peak purity, ensure that response of analyte peak in test solution is preferably equal to or less than 1 AU. If the response of API in test solution is more than 1AU, dilute the test solution accordingly and repeat the analysis. Calculate the assay and use it for mass balance study. Demonstrate the absence of interference of impurity(s) on analyte peak(s) by evaluating peak purity

v. Mass Balance: Mass balance confirms whether all degradation products are eluting during chromatographic run with suitable response. You may get lower mass balance, if you are missing few degradants or if the impurities generated have poor response as compared to API. Similarly, you may end with higher mass balance, if the impurities generated have higher response as compared to API. Mass balance for all stressed samples shall be calculated using below formula.

                                 (A+B)

Mass balance = ------------------ x 100

                                      C

Where,

A = % assay of stressed sample

B = % degradation in stressed sample (% total impurities in stressed sample - % total impurities in unstressed sample)

C = % assay of unstressed sample

Mass balance for all stressed samples shall be between 95% to 105%. If the mass balance is less than 95% or more than 105%, investigate, repeat if necessary or justify.

C. Specificity for Dissolution:

In case of dissolution, specificity must be studied for blank/diluent and placebo. Impurity interference can be studied on case to case basis.

i. Blank/Diluent Interference: Conduct interference due to diluent in case of chromatographic technique such as HPLC, GC. Inject diluent and evaluate interference at the RT of API. There must not be any interference due to diluent. Evaluation of diluent interference may not be necessary in case of spectroscopic technique, such as UV, FTIR etc.

 ii. Placebo interference: Perform dissolution as per the test method on weight of placebo equivalent to the amount present in drug product. In case of capsule dosage form use filled capsule with placebo. Analyse placebo solution and evaluate interference. In case of chromatographic technique, no interference due to placebo at the retention time of analyte shall be observed. Whereas, in case of spectroscopic technique such as UV or FTIR, placebo interference up to 2% is generally acceptable. 

iii. Impurity interference: Evaluate interference due to known and degradation products to analyte as per requirement. The impurities must not interfere with quantification of analyte. The impurity interference is not a common for dissolution specificity and it must be decided on need basis.

D. Specificity for Identification test:

In case of identification test, specificity must be studied for blank/diluent and placebo (in case of finished product such as tablets/capsule/cream etc.).

i. Blank/Diluent Interference: Conduct interference due to diluent in case of chromatographic technique such as HPLC, GC. Inject diluent and evaluate interference at the RT of API. There must not be any interference due to diluent. Evaluation of diluent interference may not be necessary in case of spectroscopic technique, such as UV, FTIR etc.

ii. Placebo interference: Perform identification test as per the test method on weight of placebo equivalent to the amount present in test solution. In case of capsule dosage form use filled capsule with placebo. Analyse placebo solution and evaluate interference. No interference due to placebo shall be observed. 

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